Biochemistry 1984, 23, 753-758 Stadel, J. M., De Lean, A., & Lefkowitz, R. J. (1980) J. Biol. Chem. 255, 1436-1441. Stadtman, T. C. (1961) Enzymes, 2nd Ed. 5, 5 5 . Stiles, G. L., Strasser, R. H., Lavin, T. N., Jones, L. R. Caron, M. G., & Lefkowitz, R. J. (1983) J . Biol. Chem. 258,
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8443-8449. Vauquelin, G., Geynet, P., Hanoune, J., & Strosberg, A. D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3710-3714. Voeikov, V., & Lefkowitz, R. J. (1980) Biochim. Biophys. Acta 629, 266-281.
Effect of Ethanol, Phenol, Formamide, Dimethyl Sulfoxide, Paromomycin, and Deuterium Oxide on the Fidelity of Translation in a Brain Cell-Free System+ Michael Laughrea,* Jacinthe Latulippe, and Anne-Marie Filion
AEISTRA~: The effects of
six different agents (ethanol, phenol, formamide, dimethyl sulfoxide, heavy water, and a misreading-inducing antibiotic, paromomycin) on the activity and the accuracy of poly(U) translation have been compared under a range (2.5-12 mM) of Mg2+ concentrations in a rat brain cell-free system. The effect of most of these agents was remarkably sensitive to the Mg2+concentration under which the assay was made. Ethanol decreased the fidelity of translation, and the efficiency of ethanol was increased 3-10-fold by higher Mg2+concentrations. The effect of paromomycin was identical with that of ethanol, despite its very different structure. Formamide, a "RNA denaturant", increased the accuracy of translation under all Mg2+concentrations tested. Dimethyl sulfoxide, another type of RNA denaturant, decreased the
accuracy of translation under all Mg2+concentrations tested. Phenol increased the accuracy of translation at high Mg2+ concentrations but decreased it at low Mg2+concentrations. D 2 0 did not change to any appreciable extent the accuracy of translation, at all the Mg2+ concentrations used. There exists a cooperativity between the effects of Mg2+and ethanol, Mg2+ and paromomycin, and Mg2+ and dimethyl sulfoxide on the fidelity of translation; no such cooperativity was detected between Mg2+ and formamide and between Mg2+and D20. The differential effects of dimethyl sulfoxide and formamide are interpreted in terms of their different dielectric constants. The dielectric constant of dimethyl sulfoxide is higher than that of water, while that of formamide is lower.
x e phenomenon of misreading mRNA has not been extensively studied in mammalian cell-free systems (Laughrea, 1981a). Nevertheless, it is widely believed that the accuracy of translation in mammalian systems is not markedly sensitive to environmental changes, in contrast to the high sensitivity of bacterial systems (Schlanger & Friedman, 1973; Burrans & Kurtz, 1977; Kurtz, 1979). This belief is implicitly based on the assumption that the effect of an environmental change (e.g., temperature, pH, organic solvents, etc.) is independent of the ionic conditions under which it is studied. In only two instances were both prokaryotic and mammalian systems compared under identical ionic conditions; the data were made largely inconclusive by the presence of a high level of endogenous RNA directed incorporation in the poly(U)-directed cell extracts (Laughrea, 1981b). In this paper, we investigated some parameters of the translation fidelity in a rat brain cell-free extract directed by poly(U). The effects of six different agents (ethanol, phenol, formamide, dimethyl sulfoxide, deuterium oxide, and a misreading-inducing antibiotic, paromomycin) on the activity and the accuracy of translation were compared under a range of Mg2+concentrations. The experimental results indicate that the effect of most of these agents is remarkably sensitive to
the Mg2+ concentration under which the assay is made, that ethanol and dimethyl sulfoxide decrease the accuracy of translation, that formamide and phenol can increase the accuracy of translation, and that the presence of deuterium oxide does not affect the fidelity of translation.
'From the Lady Davis Institute for Medical Research of the Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2. Received June 3, 1983. This work was supported by grants from the Medical Research Council of Canada and the Conseil de la Recherche en Sante du Quebec. M.L. is a recipient of a research career development award from the Conseil de la Recherche en Sante du Quebec.
0006-2960/84/0423-0753$01.50/0
Materials and Methods Animals. Male rats, either Sprague-Dawley or Fisher 344, were used. No significant difference was seen between extracts from either breed. Chemicals. Poly(U), ATP (disodium salt), GTP (disodium salt), creatine phosphate (disodium salt), calf liver tRNA, tRNA from brewers' yeast specific for phenylalanine (tRNAPhe),all enzymes, and antibiotics were purchased from Boehringer Mannheim. Paromomycin sulfate was a gift from Dr. J. J. Brossard, Parke-Davis, Canada. Spermine tetrahydrochloride and spermidine trihydrochloride were from Sigma. Deuterium oxide (D20) was purchased from Merck Sharp & Dohme, Canada. Radioactive [3H]leucine (40-60 Ci/mmol) and [ 14C]phenylalanine (>450 mCi/mmol) were purchased from both Amersham and New England Nuclear. Some of the [3H]leucine batches contained, on arrival, higher than specified levels of impurities, resulting in a doubling or even a tripling of the background incorporation. These batches were discarded or, later in the study, purified by paper chromatography using 25:4:10 1-butano1:acetic acid:water or 7:7:6 pyridine:isoamyl alcoho1:water as the solvent system. The discarded impurities were more toluene soluble than leucine itself. Most of them moved faster than leucine, but both faster moving and slower moving impurities contributed to the background incorporation. All acceptable [3H]leucinebatches 0 1984 American Chemical Society
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were used within 1 month. No impurity could be detected in the [ 14C]phenylalaninebatches. All other chemicals were of the highest purity commercially available. Phenol was redistilled before use. Buffers. Buffer A contained 7.5 mM potassium phosphate, 0.3 mM spermine, 0.4 mM spermidine, 50 mM KCl, 50 mM potassium acetate, 5 mM NH4Cl, 5 mM magnesium acetate, and 1 mM dithiothreitol, pH 7.5. The homogenization buffer was buffer A with 0.25 M sucrose20 mM N-[tris(hydroxymethyl)methyl]glycine (Tricine) (pH 7.5) and without phosphate. Cell-Free Extracts. Brains were quickly removed and rinsed in ice-cold homogenization buffer. All procedures were performed at 0-4 "C unless otherwise noted. The brains were homogenized (0.5 g wet weight/mL of homogenization buffer) with 6 strokes of a Dounce homogenizer (Bellco). The homogenate was centrifuged at 2500g for 3 min after which the supernatant was centrfuged at 20000g for 40 min. The resulting cell-free extract was preincubated for 60 min at 37 "C in homogenization buffer containing 1 mM neutralized GTP, 1 mM neutralized ATP, 10 mM creatine phosphate, 50 pg/mL creatine kinase, 50 pM amino acids, and 10 pM puromycin. This was followed by dialysis against buffer A, centrifugation for 10 min in the Eppendorf microcentrifuge, separation of the supernatants (S20) into aliquots, and storage in liquid N2. Poly( v) Translation. The basic reaction mixture contained, in 100 pL, 25 p L of S20 containing 2-3 pmol of ribosomes depending on the preparation, 5 pg of creatine kinase, 10 mM creatine phosphate, 1 mM ATP, 1 mM GTP, 7.5 mM dithiothreitol, 10 pg of unfractionated calf liver tRNA, 10 pg of tRNAPhe, 160 pg of poly(U), 10.6 pCi of neutralized [3H]leucine, 27.5 pM leucine, 0.04 pCi of [ 14C]phenylalanine, 110 pM phenylalanine, 40 p M each of all other amino acids, 1 p L of 0.1 N KOH, 7.5 mM potassium phosphate, 0.35 mM spermine, 0.45 mM spermidine, 55 mM KC1, 55 mM potassium acetate, 5 mM NH,Cl, 0.4 mM CaCl,, and from 2.5 to 12 mM magnesium acetate depending on the experiment. The final pH was 7.4. The incorporation increased linearly for more than 60 min, at which time it was stopped by the addition of 3 mL of 5% trichloroacetic acid containing 1% casamino acids. The samples were further processed as described previously (Laughrea, 1982). Care was taken to rinse the funnel of the filter holder and especially the edges of the filter after removal of the funnel. Amino acid incorporation in the absence of poly(U) was resistant to puromycin and cycloheximide, indicating that it was not ribosome directed. The backgrounds from blank samples [i.e., complete mixtures minus poly(U)] were less than 5 cpm above the machine background in the 14C channel and approximately 200 cpm above the machine background in the 3H channel. Total precipitable 3H radioactivity in the presence of poly(U) was always greater than 350 cpm and usually lower than 2500 cpm in the experiments described below. The incorporated radioactivity reported below was obtained by subtracting the results in the absence of poly(U) from the results in the presence of poly(U). Results Ethanol Decreases the Fidelity of Mammalian Translation. Figure 1 shows the effect of ethanol concentration on the fidelity of poly(U) translation in a rat brain extract. The experiment was performed at 6 mM Mg2+ and under ionic conditions which should approximate the in vivo ones (Tower, 1969; Wickson-Ginzburg & Solomon, 1963; Raina & Janne, 1975; Dratz & Handler, 1952; Rapoport, 1945). It is clear that ethanol reduced the accuracy of poly(U) translation under
LAUGHREA, LATULIPPE, A N D FILION
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